Hepatitis A virus (HAV) is a morphologically, biochemically and immunologically distinct agent which produces infectious hepatitis A in humans after an incubation period of approximately two to six weeks. Hepatitis A is a liver disease which, although not commonly fatal, can induce long periods of debilitating illness. An estimated 1.4 million cases of hepatitis A are reported annually worldwide. The disease is commonly spread by direct contact with an infected individual or by HAV-contaminated drinking water and/or food.
HAV has been characterized as a unique member of the Picornaviridae family belonging to the enterovirus group. Like other picornaviruses, HAV contains a single-stranded, positive-strand infectious RNA genome encoding a single polyprotein, which is subsequently processed into structural and nonstructural proteins.
The following four major structural capsid polypeptides of the HAV polyprotein have been described and their approximate molecular weights have been determined as follows: VP1, 30,000 to 33,000 daltons (amino acids 492–791); VP2, 24,000 to 25,000 daltons, (amino acids 24–245); VP3, 21,000 to 27,000 daltons (amino acids 246–491); and VP4, 7,000 to 14,000 daltons (amino acids 1–23).
Four major non-structural proteins have also been identified and have been designated P2A (amino acids 792–980), P2B (amino acids 981–1087), P2C (amino acids 1088–1422), P3A (amino acids 1423–1496), P3B (amino acids 1497-1519) and P3C (amino acids 1520–1738). Only one serotype appears to exist, and significant antigenic variation has not been recognized among different HAV strains.
HAV infection is typically diagnosed by the detection of IgM or IgG antibodies to the capsid proteins. Currently available recombinant proteins or synthetic peptides have not successfully been used as alternate sources of antigen in an enzyme immunoassay (EIA) format for the detection of anti-HAV, a serum marker of infection. This lack of success has been attributed to poor antigenic reactivity of the recombinant protein due to the strict conformational nature of the naturally occurring HAV antigenic epitopes. For more than 15 years, the only available source of immunoreactive proteins was from HAV cell cultures. In fact, inactivated cell culture-derived HAV is currently used by all commercial companies who manufacture anti-HAV tests. Unfortunately, HAV is made in very small quantities in cell culture, has a limited animal host range, and is difficult to purify from infected cell cultures and animal tissues. In addition to the inconvenience and cost associated with the production, purification, and standardization of cell culture-derived HAV antigen, current commercially available assays are unable to discriminate between natural infections and vaccine induced immunity as emphasized in several publications (See, e.g., Jia et al., J. Infect. Diseases 165:273-280 (1992); Robertson et al., Vaccine 10(Supp. 1):106–109 (1992); and Robertson et al., J. Med. Virol. 40:76–82 (1993)). These tests utilize intact HAV, and therefore indiscriminately detect both natural and vaccine induced immune responses.
New concepts enabling the design of efficient and inexpensive synthetic diagnostic reagents, which can be used for the development of reliable anti-HAV diagnostic tests, are necessary. As such, there remains a need in the art for synthetic peptides which can be used as alternate sources of antigen in an enzyme immunoassay (EIA) format for the detection of anti-HAV. The present invention remedies such a need.